In the autoimmune condition rheumatoid arthritis (RA), chronic inflammation reshapes cellular interactions and tissue architecture in patient joints. RA synovium is marked by expanded macrophage and fibroblast populations, extensive lymphocytic infiltration, angiogenesis and, ultimately, outgrowth beyond the natural tissue borders into cartilage and bone. In independent single-cell RNA-sequencing studies, we recently identified a novel macrophage phenotype found enriched in the synovium of RA patients (Zhang et al. Nat Immunol. 2019; Stephenson et al. Nat Comm. 2018). These macrophages express high levels of the EGF receptor (EGFR) ligand HB-EGF and are hereafter referred to as ?HBEGF+ macrophages?. Our preliminary data also demonstrated that HBEGF+ macrophages are shaped by resident fibroblast factors along with pro-inflammatory cytokines including TNF (Kuo et al. Sci Transl Med. 2019). This newly activated macrophage state then feedbacks to activate fibroblast EGFR. Our central hypothesis posits that in RA synovial tissue, HBEGF+ macrophages are polarized by fibroblasts and in turn stimulate fibroblast pathologic activity. A critical prediction is that inhibition of mediators of this disease-associated crosstalk pathway will prevent tissue remodeling. We have developed an experimental cell culture model system to study HBEGF+ macrophage differentiation and the pathologic impact of their intercellular communication with synovial fibroblasts. We have also established a patient tissue ex vivo drug response assay, which has proven effective in defining how medications function in the complex cellular interactions of inflamed synovial tissue from clinically well-defined patients. Prior reports have established the relevance of macrophage-fibroblast crosstalk as a powerful regulator of RA pathology, but these reports lacked knowledge of the precise phenotypes of the synovial macrophages in RA (Rigor). With this new information and all methods and materials in place, we can look to define which of the known pathologic tissue factors induce this macrophage phenotype, specifically testing if IFN? from abundant CD8+ T cells in the RA synovium, in combination with TNF and PGE2, induces the HBEGF+ macrophage phenotype (Aim 1). Furthermore, it is feasible to define how these macrophages drive RA synovial pathogenesis, in particular whether HB-EGF and/or epiregulin (EREG), a second EGF ligand expressed by HBEGF+ macrophages, drive both a hypoxic response and invasiveness in synovial fibroblasts and define which subtype of human synovial fibroblasts exhibit invasiveness in response to these EGF ligands (Aim 2). Finally, we are able to test the impact of potential inhibitors on HBEGF+ macrophage generation and pathologic function, focusing on the ADAM17/iRhom2 complex, which controls both the release of HB-EGF and TNF from macrophages (Aim 3). Completion of these aims will leverage our finding of human disease-associated macrophages, define mechanisms of a new pathway driving crosstalk between synovial macrophages and fibroblasts (Innovation), and lay the founding for our long- term objective of translating molecular findings to develop new therapies for the substantial number of RA patients not responding currently to therapies (Significance).